Computer algorithm for automatic allele determination from fluorometer genotyping device

ABSTRACT

The present invention provides methods and systems for an automated method of identifying allele values from data files derived from processed fluorophore emissions detected during the observation of fluorophore labeled nucleotide probes used in analyzing polymorphic DNA are provided. These methods are used in the rapid and efficient distinguishing of targeted polymorphic DNA sites without control samples.

This application is a continuation application of U.S. patentapplication Ser. No. 13/308,623, filed Dec. 1, 2011, which is acontinuation application of U.S. patent application Ser. No. 12/831,947,filed Jul. 7, 2010, which is a continuation application of U.S. patentapplication Ser. No. 11/899,199, filed Sep. 5, 2007, which is acontinuation application of U.S. patent application Ser. No. 10/085,142,filed Mar. 1, 2002. The entire contents of these applications are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of DNA genotypic analysis.More particularly, the invention relates to the allelic classificationof DNA samples through cluster analysis of analyzed emission spectraobserved from excited fluorophore-labeled nucleotide probes.Specifically, fluorophore-labeled nucleotide probes can be used verifyDNA variations between individual samples and verify the expression of aregion of DNA in different cell lines.

BACKGROUND OF THE INVENTION

Individual DNA sequence variations are known to directly cause specificdiseases or conditions, or to predispose certain individuals to specificdiseases or conditions. Such variations also modulate the severity orprogression of many diseases. Additionally, DNA sequence variationsexist between populations. Therefore, determining DNA sequencevariations is useful for making accurate diagnoses, for finding suitabletherapies, and for understanding the relationship between genomevariations and environmental factors in the pathogenesis of diseases andprevalence of conditions.

There are several types of DNA sequence variations. These variationsinclude insertions, deletions, restriction fragment length polymorphisms(“RFLPs”), short tandem repeat polymorphisms (“STRPs”), and singlenucleotide polymorphisms (“SNPs”). Of these, SNPs are considered themost useful in studying the relationship between DNA sequence variationsand diseases and conditions because they are more common, more stable,and more amenable to being employed in large-scale studies than othersorts of variations.

Currently, a set of over 3 million putative SNPs has been identified inthe human genome. It is a current goal of researchers to verify theseputative SNPs and associate them with phenotypes and diseases,eventually replacing currently-used RFLP and STRP linkage analysisscreening sets. In order to successfully accomplish this goal, it willbe necessary for researchers to generate and analyze large amounts ofgenotypic data.

A number of methods have been developed which can locate or identifySNPs. These methods include dideoxy fingerprinting (ddF), fluorescentlylabeled ddF, denaturation fingerprinting (DnF1R and DnF2R),single-stranded conformation polymorphism analysis, denaturing gradientgel electrophoresis, heteroduplex analysis, RNase cleavage, chemicalcleavage, hybridization sequencing using arrays and direct DNAsequencing.

One method of particular relevance to the present invention employs apair of fluorescent probes, each probe containing a different dye andspecific for a different allele. In this method, the two probes areadded to the DNA sample to be tested, and the mixture is amplified usingPCR. If the DNA sample is homozygous for the first allele, the firstprobe's dye will exhibit a high degree of fluorescence and thefluorescence from the second probe's dye will be absent. Conversely, ifthe DNA sample is homozygous for the second allele, the second probe'sdye will exhibit a high degree of fluorescence and the fluorescence fromthe first probe's dye will be absent. If the DNA sample is heterozygousfor both alleles, then both probes should fluoresce equally. Acommercial implementation of this method is APPLIED BIOSYSTEMS' TAQMANplatform, which employs APPLIED BIOSYSTEMS' PRISM 7700 and 7900HTSEQUENCE DETECTION SYSTEMS to record the fluorescence of each sample'sPCR product.

A typical implementation generates amplification products from a set ofa large number of samples at a time, and measures a pair of fluorescencevalues, one for each dye, from each amplified sample. To classify thesamples, it is useful to first plot the fluorescence values of theentire set on a two dimensional graph, and observe that the plottedpoints tend to cluster into separate groups according to genotype, asillustrated in FIG. 1. In this figure, a human observer can readilydiscern that the data falls into four groups. The first group, in thelower-left hand corner, represents samples that had no amplification orwere a no template control (“NTC”) reaction. The second group, in thelower right hand corner, represents those samples homozygous for Allele2. The third group, at the top, represents those samples homozygous forAllele 1. Finally, the fourth group, located between the second andthird groups, represents the heterozygous samples. This classificationis illustrated further in FIG. 2. Although it is relatively easy forhuman observer to analyze this type of data, it is necessary to developa fast, reliable, and unsupervised method of computational analysis toproduce the level of throughput necessary to analyze the large amountsof genotypic data generated.

Previous methods of computational analysis have employed a family ofalgorithms known as clustering algorithms. A typical clusteringalgorithm receives raw unstructured data and processes it to form groupsof data elements that are similar to each other. Clustering algorithmsare well known in the field of computer science, and are typicallyapplied in data mining applications. In a data mining application,clustering is used to identify relationships in data collections notreadily observable to an expert user due to the volume of information.

A typical clustering algorithm examines the distance between dataelements to find a common centroid. The centroid is mean of the value ofthe data elements belonging to a cluster. Clusters are selected by thealgorithm to minimize the distance between the elements contained withinit relative to the elements contained in other clusters. Clusteringalgorithms belong to the greater class of unsupervised machine learningalgorithms. Other supervised machine learning algorithms, includingdecision trees and neural networks, were considered for application toanalyzing output from a fluorometric genotyping device. However, allmachine learning algorithms considered were determined to beinsufficient to analyze this type of data accurately. A thorough reviewof initial collection of 80 human reviewed outputs revealedcharacteristics of the data that would not allow standard machinelearning algorithms to work with a high degree of accuracy.

It is an object of this invention to provide a fast, accurate, andunsupervised method of classifying genotypic samples based onfluorometric data generated from them.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for categorizing themembers of a dataset into discrete categories. In this aspect thedataset has a plurality of datapoints, and each datapoint has at leasttwo numerical values associated with it. In this aspect, the method hasthe following steps: Assign each datapoint an angular value based onthat datapoint's numerical values; sort the dataset by angular value;calculate the differences between adjacent angular values in the sorteddataset; determining category-dividing values by identifying differencesthat are larger than a predetermined threshold; and classifyingdatapoints according to their angular values relative to thecategory-dividing values.

In a further aspect, each datapoint has exactly two numerical values,and the angular value is an arctangent of the datapoint's numericalvalues. In a further aspect the numerical values are normalized beforethe angular values are calculated.

In a further aspect, the numerical values represent fluorometric data,wherein the different numerical values for each datapoint represent thefluorescence of a different dye.

In a further aspect, the method identifies exactly two category-dividingvalues and three categories. In a further aspect, these three categoriesrepresent homozygosity for a first allele, homozygosity for a secondallele, and heterozygosity for both alleles.

In a further aspect the fluorometric data is measured from the productof an amplification reaction, and the method includes a step forremoving datapoints that represent either a control reaction or afailure to amplify. In this aspect, the datapoints whose Euclideandistance falls beneath a predetermined threshold are removed from anyfurther classification.

In a further aspect, the results of the classification are examined todetermine whether to bring them to the attention of a human user. Inthis aspect, the results are examined for conditions that indicate thatthe classification was unsuccessful. Such conditions include excessclassification in one category, classification into more than threecategories, absence or near absence of any classification in one or morecategories, unclassified datapoints, inadequate separation from controlor nonamplification reactions, clusters having angular values that areeither too high or too low, clusters whose ranges of angular values aretoo wide, classification that is not compatible with a Hardy-Weinbergequilibrium, and control or nonamplification reactions that are too farfrom the origin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a two dimensional scatterplot of fluorometric data.

FIG. 2 provides a two dimensional scatter plot of fluorometric dataclassified by allele.

FIG. 3 provides a two dimensional scatterplot of raw fluorometric data.

FIG. 4 provides a two dimensional scatterplot of normalized fluorometricdata.

FIG. 5 provides a two dimensional scatterplot of normalized fluorometricdata classified by allele.

FIG. 6 provides a two dimensional scatterplot of normalized fluorometricdata classified by allele, and undetermined datapoints identified.

FIG. 7 provides a bargraph of the differences in arctangent valuesbetween adjacent datapoints sorted by arctangent.

DETAILED DESCRIPTION OF INVENTION

Although the methods of the current invention can be used to classifyany kind of bivariate or multivariate data, they are particularly usefulfor classifying genotypic data, especially allelic data generated byfluorescence.

In one embodiment, the fluorometric genotyping device generates for eachsample a unique sample identifier, a value from one of the sample'sfluorometric probes, and a value from the sample's other fluorometricprobe. The paired fluorescence values can then be plotted as x and yvalues on a two-dimensional grid. Ideally, the data generated in thisfashion should yield heterozygous datapoints in the upper-rightquadrant, and homozygous datapoints for each allele in the upper-leftand lower-right quadrants, respectively. If that were the case, thenallele calling would be a simple matter of dividing the grid byquadrants. However, fluorometric genotypic has been observed to exhibitseveral characteristics and idiosyncrasies that must be addressed inorder for an automated allele caller to function accurately, and havenot been adequately addressed by previous methods of clustering andmachine learning.

Traditional machine learning algorithms such as decision trees andneural networks generate solutions by training on a given collection ofdata where the outcome is known and then applying the trained system topredict data in unknown outcomes. Training these algorithms withfluorometric data produces a solution where a set of X and Y boundariesare defined that had the highest probability of being correct. However,the predictions of results that deviate from the trained form would havepoor accuracy. Because fluorometric data tends to vary from instance toinstance, a trained system is too rigid to be sufficiently accurate tooperate unsupervised on a large number of samples.

Another imperfection of fluorometric data is that outputs fromfluorometric genotyping devices can validly produce one to threeclusters, in addition to clusters formed by samples that had noamplification or no template control reactions (“NTCs”). In general,fluorometric genotyping devices are expected to produce three clusters,but can validly produce only one or two. For example, if three clustersare expected but only two valid clusters are produced, then thedatapoints could be invalidly categorized into three clusters.Clustering algorithms are generally given a fixed number of expectedclusters, and any deviation from the expected number of clusters greatlyreduces their effectiveness.

Another imperfection of fluorometric data is that datapoints belongingto the same category can be spread out spatially. A widely-spreadcluster is usually observed for the category of heterozygous genotypes,where both fluorometric probes are active. A widely-spread clustergreatly reduces the effectiveness of clustering algorithms, especiallywhen the distance between the furthest datapoints of a cluster and itscentroid is greater than their distance to other clusters. Furthermore,as noted above, the number of valid clusters produced by fluorometricdata can vary from the expected three to one or two. For example if twovalid clusters are produced and one of them is widely-spread, it islikely that a clustering algorithm will incorrectly divide that onevalid cluster into two invalid clusters.

In one embodiment, the dataset has a plurality of datapoints, and eachdatapoint has two numerical values associated with it. In an alternateembodiment, each datapoint has more than two numerical values associatedwith it. In a further embodiment, the numerical values representquantitative empirical data. In yet a further embodiment, thequantitative empirical data is measured fluorescence. In a furtherembodiment, the numerical values are normalized before being used in anysubsequent calculations.

In this embodiment, an angular value is calculated for each datapoint inthe dataset based upon the datapoint's numerical values. In a furtherembodiment, the angular value is an arctangent of the numerical values.The dataset is then sorted by angular value. A difference value is thencalculated for each datapoint by subtracting the angular value of theprevious datapoint from that of the current datapoint. The differencevalue of the first datapoint is that point's angular value.

If the difference value is large enough to exceed a predeterminedthreshold, a new category-dividing value is designated between the twoangle values from which that difference value was calculated. In oneembodiment, the category-dividing value is the average of the two anglevalues from which the above-threshold difference value was calculated.FIG. 7 illustrates the difference values for an example dataset. In thisexample, the samples are lined along the X-axis according to the rank oftheir angular value, and each sample's difference value is plotted onthe Y-axis. As illustrated in FIG. 7, the results indicate the presenceof two difference values which stand out dramatically from the rest ofthe data. Two dividing values are designated, one between sorted samples131 and 132 and the other between sorted samples 222 and 223, each at aangle value between the two angle values which generated theabove-threshold difference value.

As their name suggests, the category-dividing values are subsequentlyused to separate datapoints into categories. In this example, sorteddatapoints 1-131 are classified as homozygous for a first allele, sorteddatapoints 132-222 are classified as heterozygous, and samples 223-239are classified as homozygous for a second allele.

The data contained in the example illustrated in FIG. 7 and describedabove are relatively clean and well-adapted to machine analysis. In oneembodiment, the data are examined for conditions which indicate that thedata are less well-formed and may not yield correct results whensubjected to unsupervised machine analysis. In a further embodiment, ifsuch conditions are detected, the method adapts its analysis to theidiosyncrasies of the dataset in order to yield a more accurateanalysis. In yet a further embodiment, if such conditions are detectedthe dataset is flagged to indicate that it should be examined by a humanreviewer.

In one embodiment, the data are examined to determine if control samplesare present in the dataset. In a further embodiment, identified controlsamples are removed from the dataset before any further classificationis performed.

In one embodiment, the range between the maximum and minimum observedvalues for each fluorophore-labeled nucleotide probe is calculated. Ifthe range falls below a predetermined threshold, it is determined thatthe results are only valid for the other probe. Those samples producingdata with the valid probe are then distinguished from samples that hadno amplification or were NTCs. In one embodiment, all datapoints withina predetermined distance from the minimum observed values of the datasetare determined to be NTCs and the remaining datapoints are classified asbelonging to the observed probe.

If, on the other hand, the range between maximum and minimum observedvalues for each fluorophore-labeled nucleotide probe exceeds thepredetermined threshold, it is determined that multiple clusters areprobably present, and the following steps are taken: All of thenumerical values are normalized. In a further embodiment, all of thenumerical values are normalized on a scale ranging from 0.0 to 1.0. Thenthe Euclidean distance between minimum values and each sample iscomputed. Samples are predicted as NTC or non-amplification and removedfrom further consideration if their distance to minimum values fallbelow a predetermined distance threshold.

The average distance of all remaining datapoints is then computed andused to calculate a threshold. All remaining datapoints that fall belowthis threshold are predicted as undetermined and removed from furtherclassification.

Once the above-described screening steps are performed, the method ofthis embodiment proceeds similarly to that of the previous example:Angular values are calculated for each datapoint; the dataset is sortedby angular value; difference values are calculated; category-dividingvalues are identified; and each datapoint is categorized according toits angular value.

In one embodiment, the classification results are then examined with aseries of evaluations to determine if there are any characteristics tobring to the attention of a human reviewer. Examples of such conditionsinclude excess classification in one category, classification into morethan three categories, absence or near absence of any classification inone or more categories, unclassified datapoints, inadequate separationfrom control or nonamplification reactions, clusters having angularvalues that are either too high or too low, clusters whose ranges ofangular values are too wide, classification that is not compatible witha Hardy-Weinberg equilibrium, and control or nonamplification reactionsthat are too far from the origin.

If the samples were identified as all homozygous, it not considered anerror of the clustering algorithm, but needs to be noted to theinvestigator that the assay is not variable.

If only one cluster was identified and it could not be determined to beall homozygous then the dataset is flagged to indicate that human reviewis recommended.

If more than three clusters were identified then the dataset is flaggedso that a human user can review the calls.

If more than a preset number of datapoints are predicted as undeterminedthen the dataset is flagged indicating that human review is desired. Ina further embodiment, this preset number is 4.

If the samples from a probe are not separated from the node templatecontrols by a threshold distance determined by the probe technology usedthen the dataset is flagged to indicate that the probe is producing aweak signal.

If the heterozygosity of the predicted calls is greater than a givenheterozygosity threshold then the dataset is flagged so that a humanuser can review the predicted results. Heterozygosity is the predictednumber of heterozygous sample divided by the number of heterozygous andhomozygous samples.

If the homozygous cluster for a first allele is below an arctangent of1.0 then the dataset is flagged indicating that the cluster is in toolow of a position and should be human reviewed.

If the homozygous cluster for a second allele is above an arctangent of0.67 then the database is flagged indicating that the cluster is in toohigh of a position and should be human reviewed.

If the heterozygous cluster is above an arctangent of 1.35 then thedatabase is flagged indicating that the cluster is in too high of aposition and should be human reviewed. Similarly, if the heterozygouscluster is below an arctangent of 0.18 then the database is flaggedindicating that the cluster is in too low of a position and should behuman reviewed.

If there are three clusters and the cluster with the smallest number ofsamples, also known as the minor allele cluster, is greater then theheterozygous cluster then these results do not agree with populationgenetics Hardy-Weinberg principle and the database is flagged so that ahuman will review the results.

If any cluster is wider then 0.6 from the start of the cluster'sarctangent to its end then the cluster is unusually wide and the datasetis flagged to have the results human reviewed.

If the center of the predicted node template control cluster is greaterthen 0.3 on a probe axis in a 0.0 to 1.0 normalized coordinate system,this indicates a problem with the probe and the dataset is flagged sothe results are human reviewed.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrative,rather than limiting, of the invention described herein. Scope of theinvention is thus indicated by the appended claims, rather than by theforegoing description, and all variants which fall within the meaningand range of equivalency of the claims are therefore intended to beembraced therein.

What is claimed:
 1. A method for automatic allele detection by amachine, the method comprising: operating a fluorometric detectioninstrument on a plurality of biological samples that have been subjectto an amplification assay, wherein the amplification assay comprisesusing, with each sample of the plurality of samples, a firstfluorometric probe that probes for presence of a first allele and asecond fluorometric probe that probes for presence of a second allele,wherein operating the fluorometric detection instrument on the pluralityof biological samples generates a plurality of light measurements, eachlight measurement comprising, with respect to a sample of the pluralityof samples, a numerical fluorescence value associated with the firstfluorometric probe and a numerical fluorescence value associated withthe second fluorometric probe; generating a plurality of angular valuesby determining an angular value for each light measurement; sorting thelight measurements in a sequence ordered by the angular values;generating a plurality of difference values by calculating differencesbetween the angular values adjacent to each other in the sequence;determining at least one category-dividing value by identifying at leastone of the difference values above a predetermined threshold gap; andobtaining a plurality of classification results of samples correspondingto the light measurements by classifying each light measurementaccording to a corresponding angular value relative to thecategory-dividing value to obtain at least one classification result fora corresponding amplified sample, the classification result identifyingthe corresponding amplified sample as either homozygous with respect tothe first allele, homozygous with respect to the second allele, orheterozygous with respect to the first and second alleles.
 2. The methodof claim 1 wherein each light measurement comprises two numericalvalues, each a fluorescence value from a different fluorometric probe.3. The method of claim 2 wherein the angular value is an arctangent ofthe two numerical values.
 4. The method of claim 1 wherein the numericalvalues are transformations of fluorometric properties.
 5. The method ofclaim 1, further comprising determining two category-dividing valuescomprising a first category-dividing value and a secondcategory-dividing value.
 6. The method of claim 1 further comprisingnormalizing the numerical values to a scale.
 7. The method of claim 6,wherein the scale ranges from 0.0 to 1.0.
 8. The method of claim 1further comprising: excluding non-amplification light measurements frominfluencing the classification result by: (i) calculating a Euclideandistance for each light measurement; and (ii) excluding selected lightmeasurements from influencing the classification result when a Euclideandistance of the selected light measurement falls below a predetermineddistance threshold.
 9. The method of claim 5 wherein classifying atleast one of the light measurements further comprises: classifying atleast one first light measurement in a first category, wherein theangular value of the at least one first light measurement is lower thanthe first category-dividing value and the second category-dividingvalue; classifying at least one second light measurement in a secondcategory, wherein the angular value of the at least one second lightmeasurement is between the first category-dividing value and the secondcategory-dividing value; and classifying at least one third lightmeasurement in a third category, wherein the angular value of the atleast one third light measurement is greater than the firstcategory-dividing value and the second category-dividing value.
 10. Themethod of claim 9 wherein classification in the first categorycorresponds to homozygosity for the first allele, classification in thethird category corresponds to homozygosity for the second allele, andclassification in the second category corresponds to heterozygosity forthe first and second alleles.
 11. The method of claim 1 furthercomprising: determining, based on the plurality of classificationresults, whether a condition is present requiring review by a humanuser; and if the condition is present, flagging the plurality ofclassification results for human user review.
 12. The method of claim 11wherein: the condition comprises a proportion of light measurementsclassified as heterozygous exceeding a predetermined threshold.